1. Overview

GST fusion protein purification magnetic beads (Magarose-GSH) are a new purification medium developed by PuriMag for the purification of GST-tagged fusion proteins, which can directly obtain high-purity target proteins from biological samples in one step through magnetic separation, greatly simplifying the purification process and improving purification efficiency, and is suitable for convenient purification of GST fusion proteins in scientific research and industry. GST pull-down experiments using Magarose-GSH beads are simple and fast, offering advantages that traditional methods cannot match.

2. Product characteristics

Product Name	Magarose-GSH
Item number	PMAG002
Magnetic bead concentration	25%(v/v)in 20% ethanol
ligand and density	20-30 umol GSH/ml magnetic beads
Medium	6% cross-linked magnetic agarose
Particle size	20-45μm
Ligand binding	6-10 mg GST fusion protein/ml magnetic beads
Save	Store at 2~8°C for one year

3. How to use

1. GST-tagged protein purification

Binding/eluent: 140 mM NaCl, 2.7 mM KCl, 10 mM_Na2 HPO4, 1.8 mM KH 2 PO ₄, pH 7.4；

Eluent: 50 mM Tris-HCl, 50 mM reduced glutathione, pH 8.0; Eluent preparation: 50 mL of 0.1 M Tris solution, 1.535 g reduced glutathione, then pH adjusted to 8.0 with 0.1 M hydrochloric acid and deionized water to 100 mL.

The magnetic bead usage is calculated by the user based on the target protein yield and magnetic bead load information, here is only an example of obtaining 5-10 mg of target protein:

1.1 Magnetic bead preparation: Mix the GST purified magnetic beads thoroughly, use a pipette to take 4 ml of magnetic bead suspension (magnetic bead volume is 1 ml) and put it in the centrifuge tube, put the centrifuge tube on the magnetic separator, and after the solution is clarified, use the pipetting solution and add deionized water to wash and remove the alcohol repeatedly.

1.2 Magnetic bead balance: Add the same volume of binding/washing solution as the suspension, blow repeatedly with the gun tip 5 times, place the centrifuge tube on the magnetic separator, wait for the solution to be cleared, remove the clear solution, and wash twice repeatedly.

1.3 Magnetic beads bind to proteins: Add GST fusion protein lysate to the treated magnetic beads, mix upside down, and incubate at room temperature for 30 minutes (if the target protein is unstable, it is recommended to incubate at 2-8 °C for 1 h).

1.4 Magnetic bead washing: Place the centrifuge tube in the magnetic separator, and after the solution becomes clear, transfer the supernatant to a new centrifuge tube and keep it for testing. Add 2 times the volume of suspended liquid to the centrifuge tube, blow repeatedly 5 times with the spear tip, place the centrifuge tube in the magnetic separator, wait for the solution to be cleared, transfer the supernatant to a new centrifuge tube, and keep it for testing. Repeat the impurity washing step 2 times.

1.5 Elution: Add the eluent of the same volume as the suspension, blow it 5 times with a pipette, incubate at room temperature for 5-10 minutes, put it on a magnetic separator, and after the solution is clarified, aspirate the supernatant, which is the target protein component. The above steps were repeated several times to collect the eluting components and leave samples for testing to increase the recovery of the protein of interest.

1.6 SDS-PAGE detection: The purified samples will be detected by SDS-PAGE.

Note: 1. Glutathione is used in the ready.

2. For most GST fusion proteins, the target protein can be eluted with an eluent containing 10mM reducing glutathione. For a small number of GST fusion proteins with strong binding ability, the elution time can be appropriately extended, the number of elutions can be increased, or the concentration of reduced glutathione in the eluent can be increased.

3. 1~5mM EDTA, 1~10mM DTT, 0.1~1.0%Triton X-100, 0.1~1.0%Tween 20, etc. can be added to the binding/eluent and eluent.

1.7 Magnetic bead cleaning and preservation: After simple cleaning and treatment, the magnetic beads can continue to be used for subsequent purification operations and can also be stored for a long time. Users can choose the cleaning method according to the use of magnetic beads, mainly including:

Case 1: When the number of reuses is small and the binding capacity is not obvious, it can be cleaned by alternating high pH and low pH buffers

w High pH wash (alkaline wash): Add 10 mL of wash solution A (0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5) to the used magnetic beads, swirl for 60 s, magnetically separate, and remove the supernatant;

w Low pH wash (pickling): add 10 mL of wash solution B (0.1 M sodium acetate, 0.5 M NaCl, pH 4.5), vortex shaking for 60 s, magnetically separate, and remove the supernatant;

w Repeat alkaline washing and pickling alternate cleaning 2 times, a total of 3 times.

Situation 2: When the number of reuses is high, the ability of magnetic beads to bind the target protein is significantly reduced due to precipitation, denaturation, or accumulation of non-specific adsorbed proteins. To remove precipitated or denatured proteins, wash them as follows:

w Wash twice with 5 mL of 6 M guanidine hydrochloride for 60 s each time, magnetically separate, and remove the supernatant.

Wash 3 times with 10 mL of 1X PBS for 60 s each whirlpool, magnetically separate, and remove the supernatant.

Case 3: To remove the hydrophobic binding substances, you can wash them as follows:

w Wash 3 times with 5 mL of 70% ethanol or 0.1% non-ionic surfactant for 60 s each time, magnetically separate, and remove the supernatant.

Wash 3 times with 10 mL of 1X PBS for 60 s each time, magnetically separate, and remove the supernatant.

Note: After completing the cleaning of cases 1 or 2, if the user needs to continue to use magnetic beads to purify the protein, it needs to be washed with binding/washing solution 2~3 times first. If you do not need to continue to use it, wash the beads with 20% ethanol 2~3 times, and then add 20% (v/v) ethanol to the beads to make the total volume 4 mL, and store at 2~8°C.

2．Pull-down operation process

Binding/washing Buffer: 140 mM NaCl, 2.7 mM KCl, 10 mM_Na2 HPO4, 1.8 mM KH 2 PO ₄, pH7.4

Elution Buffer: 50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0

1) Magnetic bead preparation: take an appropriate amount of GST purified magnetic beads (the volume of magnetic beads is 10% of the total volume), turn the magnetic beads upside down repeatedly, mix them well, use a pipette to take an appropriate amount of magnetic bead suspension, put it in a centrifuge tube, put the centrifuge tube in a magnetic separator, and aspirate the clear solution after the solution is clarified.

2) Magnetic bead balance: remove the centrifuge tube from the magnetic separator, add the same volume of binding solution as the suspension, blow repeatedly 5-10 times, put the centrifuge tube on the magnetic separator, wait for the solution to be clear, suck out the clear solution, and wash twice.

3) Magnetic beads bind target proteins: GST-tagged target protein samples are added to the treated magnetic beads and mixed upside down. Place the centrifuge tube on the mixer and incubate at room temperature for 30 minutes.

4) Magnetic bead impurities: Place the centrifuge tube in a magnetic separator, and after the solution is clarified, remove the supernatant and keep it for sampling and testing. Add 2 times the volume of suspended liquid to the centrifuge tube, blow repeatedly 5-10 times, place the centrifuge tube on the magnetic separator, and after the solution becomes clear, aspirate the supernatant, and repeat the above steps twice to obtain the target protein-magnetic bead complex.

5) Binding of the target protein to the target protein-magnetic bead complex: Samples containing the target protein are added to the treated target protein-magnetic bead complex and mixed upside down. Place the centrifuge tube on the mixer and incubate at room temperature for 30 minutes.

6) Magnetic bead impurities: Place the centrifuge tube in the magnetic separator, and after the solution is clarified, remove the supernatant and keep it for sampling and testing. Add 2 times the volume of suspended liquid to the centrifuge tube, blow repeatedly 5-10 times, place the centrifuge tube on the magnetic separator, and aspirate the supernatant after the solution becomes clear. Repeat the above steps 2 times. The protein of interest is captured from the mixed system by binding to the target protein.

7) Elution of the target protein: add the eluent of the same volume as the suspension to the above centrifuge tube, blow with a pipette 5 times, then put it in the flip mixer or manually turn the centrifuge tube gently at room temperature, after 5-10 minutes, put it on the magnetic separator, and after the solution becomes clear, aspirate the supernatant and collect the eluting components, that is, the target protein and the target protein complex are obtained. It can also be repeated again to collect the eluting components separately and leave them for testing.

3. Precautions

1) Freezing, drying, and high-speed centrifugation should be avoided during the use and storage of magnetic beads.

2) Before using this product, please be sure to fully oscillate the magnetic beads to maintain a uniform suspension state;

3) During the mixing process of magnetic beads and solution, if the solution is viscous and cannot be resuspended by flipping the centrifuge tube, the magnetic beads can be fully resuspended by repeated blowing with a pipette or by swirling for a short time;

4) Users can retain the supernatant removed by magnetic separation for sampling and testing according to actual needs, so as to analyze the purification process and optimize the protein purification process;

5) This product can be reused, when reusing, it is recommended to purify the same protein, and when purifying different kinds of protein, it is recommended to use new magnetic beads to prevent cross-contamination;

6) This product needs to be used with a magnetic separator;

This kit can only be used for in vitro experiments, not for clinical, therapeutic and in animal experiments, etc., and is not responsible for the consequences arising therefrom.